Genetic Studies Of Rotavirus Pathogenesis And Development Of Rotavirus Vaccines

轮状病毒发病机制的遗传学研究和轮状病毒疫苗的开发

• 批准号: 7592135
• 负责人: Yasutaka Hoshino
• 金额: $ 18.29万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592135
• 关键词: Baculoviruses; Bos taurus; Capsid; Cattle; Cercopithecus pygerythrus; Colostrum; Cultured Cells; Development; Diarrhea; Drug Formulations; Enterotoxins; Family suidae; Gastrointestinal tract structure; Genes; Genetic; Gnotobiotic; Goals; Human; Human Herpesvirus 4; Infection; Macaca mulatta; Modeling; Mus; Mutation; Newborn Infant; Oral; Pathogenesis; Play; Proteins; Recombinants; Role; Rotavirus; Rotavirus Infections; Rotavirus Vaccines; Sampling; Serial Passage; Serotyping; Sus scrofa; System; Time; Vaccines; Viral; Viral Genes; Virulence; Virulent; Virus; Virus Replication; attenuation; base; day; design; kidney cell; mouse model; pup; tissue tropism

Previously, in studies involving a semi-homologous system of gnotobiotic newborn pigs and a virulent porcine rotavirus strain (SB-1A) and an avirulent human rotavirus strain (DS-1) and their reassortants, we demonstrated that: (i) the third (VP3), fourth (VP4), ninth (VP7), and tenth (NSP4) porcine rotavirus gene each play an important independent role in the virulence of rotavirus infection in piglets; and (ii) all four of the porcine rotavirus virulence-associated genes are required for the induction of diarrhea and the shedding of rotavirus by piglets. These observations suggested a potential new strategy for attenuation of wild-type human rotaviruses of major epidemiologic importance and its application to the development of a safe and effective vaccine. Previously, we developed rhesus (RRV)-, bovine (UK)-, and porcine (Gottfried)-based multivalent vaccine candidates which were designed to provide antigenic coverage for VP7 (G) serotypes 1-4, 8 and 9 of epidemiologic importance. This year, in order to study which gene(s) of a murine rotavirus EB strain are involved in induction of diarrhea in a homologous mouse model, we passaged a cell culture-adapted avirulent (non-diarrheagenic) murine rotavirus EB strain serially in 4-5 day-old CD-1 mice until the virus became virulent (diarrheagenic). This virulent virus was then inoculated onto primary African green monkey kidney (AGMK) cells and passaged serially 18 times. The 18th cell culture-passaged virus that was shown to be avirulent in mouse pups became virulent again during subsequent serial passage in CD-1 mice. This virulent virus when passaged again serially 18 times in primary AGMK cells became avirulent in mice. Sequencing of all 11 genes of selected virulent and avirulent viruses generated in this study, revealed that virulent viruses when compared with avirulent viruses bore (i) distinct mutations in gene 4 (encoding outer capsid VP4) and in gene 10 (encoding viral enterotoxin NSP4) and (ii) no significant mutations in other genes. In addition, no significant difference in virus replication efficiency in mice was observed between virulent and avirulent viruses. We generated 3 baculovirus recombinants expressing the NSP4 protein derived from high mouse-passaged virulent EB virus (sample A), low mouse-passaged virulent EB virus (sample B), or cell culture-passaged avirulent EB virus (sample C). Upon oral inoculation in mouse pups, the development of diarrhea was observed in 91.6% (11 of 12) in sample A group; 12.5% (1 of 8) in sample B group; and 0% (0 of 12) in sample C group, indicating that the NSP4 protein played an important role in pathogenesis in this model.

此前，在涉及生猪轮状病毒强毒株(SB-1A)和人轮状病毒弱毒株(DS-1)及其重组体的半同源系统的研究中，我们证明：(I)猪轮状病毒第三(VP3)、第四(VP4)、第九(VP7)和第十(NSP4)基因分别在仔猪轮状病毒感染的毒力中发挥重要的独立作用；以及(Ii)所有四个与猪轮状病毒毒力相关的基因都是诱导仔猪腹泻和轮状病毒脱落所必需的。这些观察结果提示了一种潜在的新策略，用于减弱具有重要流行病学意义的野生型人类轮状病毒，并将其应用于安全有效的疫苗的开发。此前，我们开发了基于恒河猴(RRV)、牛(UK)和猪(Gottfred)的多价疫苗候选疫苗，旨在为具有流行病学重要性的VP7(G)血清1-4、8和9型提供抗原覆盖。今年，为了研究小鼠轮状病毒EB株的哪些基因(S)参与了同源小鼠模型的腹泻诱导，我们在4-5日龄的CD-1小鼠中连续传代了一株细胞培养适应的无毒(无腹泻)小鼠轮状病毒EB株，直到病毒变得强毒(致腹泻)。然后将该毒株接种到原代非洲绿猴肾(AGMK)细胞上，连续传代18次。第18代细胞培养传代的病毒在小鼠幼鼠中被证明是无毒的，在随后的CD-1小鼠的连续传代中再次成为强毒。这种毒力强的病毒在原代AGMK细胞中再次连续传代18次后，在小鼠身上变得无毒。对本研究中产生的所有11个强毒和无毒病毒的基因进行了测序，结果表明，强毒与无毒病毒相比，(1)基因4(编码外衣壳VP4)和基因10(编码病毒肠毒素nsp4)有明显的突变，(2)其他基因没有显著突变。此外，强毒和无毒病毒在小鼠体内的病毒复制效率没有显著差异。我们构建了3个表达NSP4蛋白的杆状病毒重组体，分别来自高小鼠传代的强毒EB病毒(样本A)、低小鼠传代的强毒EB病毒(样本B)和细胞培养传代的无毒EB病毒(样本C)。小鼠口服接种后，样本A组腹泻发生率为91.6%(11/12)，样本B组为12.5%(1/8)，样本C组为0(0/12)，说明NSP4蛋白在该模型的发病机制中起重要作用。